Early forming oligomers of the amyloid beta peptide are believed to play a critical role Alzheimer's disease (AD) by binding to, and permeabilizing, biological membranes thus being toxic to neurons. However, understanding is still lacking of which of the many Abeta species observed are cytotoxic in vivo, what is the underlying mechanism for toxicity, how do the toxic aggregates form, how do they interact with cellular membranes, why the pathology specific to neurons and why do the interactions depend on the cell's age (i.e., what is the molecular/cellular basis for the age-relatedness of AD?). These are experimentally challenging problems since the Abeta oligomers are highly heterogeneous (their sizes range from dimers to hundreds of peptide monomers) and are also highly metastable and therefore exist only transiently. As a result, studies of the Abeta oligomers by traditional approaches that use large ensembles of molecules and relatively high peptide concentrations, are not only of limited physiological significance, but also inherently hampered by the fact that ensemble-averaging masks low amounts of transient intermediates and cannot effectively resolve their dynamic heterogeneity. Our long-term goal is to contribute to the understanding of Abeta`s cellular toxicity by applying single molecule spectroscopy (SMS) to investigate the formation and interaction of individual peptide oligomers with cultured neuronal cells. Characterizing the interactions of single cell-attached oligomers in real-time will allow us to identify the toxic species and to quantify their cell permeabilization efficacy. Extending SMS to the study of peptide-induced membrane disruption in cells as complex as neurons will also create a major advance in SMS and membrane biophysics. This new capability will allow us to address the following two hypothesis-driven aims: Aim 1. To adapt the SMS approach to the study of Abeta interaction with cultured neurons and to test the hypothesis that the predominant mechanism for the formation of peptide oligomers at A[unreadable] concentrations typical for brain tissue, is by assembly of membrane-bound peptide species. We will apply SMS to monitor the time evolution of individual Abeta oligomeric species on the surface of cultured neurons. Aim 2. To test the hypothesis that permeabilization of the neuronal membrane by Abeta is highly dependent on oligomer type and on its localization on the cell surface. Imaging single neurons loaded with a fluorescent calcium indicator, we will use SMS to monitor individual Abeta oligomers on the membrane, as in Aim 1 above, and simultaneously record (at a second wavelength, where the Ca indicator emits) permeabilization events, the location of each pore on the cell surface and quantify its permeabilizing efficacy via the intensity of calcium influx associated with it. We will then follow the time evolution of calcium leakage through each individual pore to derive, for example, a dynamic trace of the evolution of pore size. The results will also allow us to determine whether specific domains on the neuron are more susceptible to oligomer formation/permeabilization allowing, in future studies, to identify the origin of the specificity. PUBLIC HEALTH RELEVANCE: The main etiology of Alzheimer's disease (AD) is the loss of nerve cells in certain areas of the brain. There is strong evidence that this is due to toxic complexes formed by a peptide termed amyloid beta. The current study will explore the molecular interactions that lead to the formation of these toxic structures on the cell surface and the mechanism by which they exert their toxicity. This basic knowledge will enhance our understanding of AD and can serve in the future for the design of intervention strategies for the disease.